Lam-Yuk-Tseung, S., Picard, V., and Gros, P., “Identification of a tyrosine-based motif (YGSI) in the amino terminus of Nramp1 (Slc11a1) important for lysosomal targeting”, Journal of Biological Chemistry, 281(42):31677-88, 2006.
IdentificationofaTyrosine-basedMotif(YGSI)intheAmino
TerminusofNramp1(Slc11a1)ThatIsImportant
forLysosomalTargeting
*□S
Receivedforpublication,February27,2006,andinrevisedform,July25,2006Published,JBCPapersinPress,August12,2006,DOI10.1074/jbc.M601828200
StevenLam-Yuk-Tseung,VirginiePicard,andPhilippeGros1
FromtheDepartmentofBiochemistry,McGillCancerCenterandCenterforHostResistance,McGillUniversity,Montreal,
QuebecH3G1Y6,Canada
Inmacrophages,Nramp1(Slc11a1)isexpressedinlysosomes
andrestrictsreplicationofintracellularpathogensbyremoving
divalentmetals(Mn
2andFe2)fromthephagolysosome.
Nramp2(DMT1,Slc11a2)isexpressedbothattheduodenal
brushborderwhereitmediatesuptakeofdietaryironandubiq-
uitouslyattheplasmamembrane/recyclingendosomesofmany
celltypeswhereittransportstransferrin-associatedironacross
theendosomalmembrane.InNramp2,acarboxyl-terminal
cytoplasmicmotif(
555YLLNT559)iscriticalforinternalization
andrecyclingofthetransporterfromtheplasmamembrane.
Herewestudiedthesubcellulartraffickingpropertiesof
Nramp1andinvestigatedthecis-actingsequencesresponsible
fortargetingtolysosomes.Forthis,weconstructedandstudied
Nramp1/Nramp2chimericproteinswherehomologous
domainsofeachproteinwereexchanged.Chimerasexchanging
theamino-(upstreamTM1)andcarboxyl-terminal(down-
streamTM12)cytoplasmicsegmentsofbothtransporterswere
stablyexpressedinporcineLLC-PK
1kidneycellsandwerestud-
iedwithrespecttoexpression,maturation,stability,cellsurface
targeting,transportactivity,andsubcellularlocalization.An
Nramp2isoformIIchimerabearingtheaminoterminusof
Nramp1wasnotexpressedatthecellsurfacebutwastargetedto
lysosomes.Thislysosomaltargetingwasabolishedbysingleala-
ninesubstitutionsatTyr
15andIle18ofa15YGSI18motifpresent
intheaminoterminusofNramp1.TheseresultsidentifyYGSIas
atyrosine-basedsortingsignalresponsibleforlysosomaltarget-
ingofNramp1.
Nrampdefinesalarge,highlyconservedfamilyofintegral
membraneproteinsthattransportdivalentmetalsinapH-de-
pendentfashion(1).Thefirstmemberofthisfamily,Nramp1
(Slc11a1),wasidentifiedbypositionalcloningofalocus
(Bcg/Ity/Lsh)thatregulatessusceptibilityofmicetoinfec-
tionwithanumberofunrelatedintracellularpathogens(2).
Naturallyoccurring(Nramp1
G169D)orexperimentally
induced(Nramp1/)loss-of-functionmutationsatNramp1
causesusceptibilitytoinfectionwithseveralspecies
ofMycobacterium,Salmonella,andLeishmania(3,4).Poly-
morphicvariationsathumanNRAMP1havealsobeenlinked
toincreasedsusceptibilitytotuberculosisandleprosyin
areaswherethosediseasesarethemostprevalent(5–7).
Hydropathyprofilingandtopologicalstudiessuggestthat
Nramp1iscomposedof12putativetransmembrane
domainsandisexpressedprimarilyinlysosomesofmono-
nuclearphagocytesandintertiarygranulesofpolymorpho-
nuclearleukocytes(1).Uponphagocytosisofinertparticles
oroflivepathogens,Nramp1israpidlyrecruitedtothe
membraneofmaturingphagosomes(8–11).Recentwork
hasshownthatNramp1functionsasaphagosomalmetal
effluxpumpthattransportsdivalentcationssuchasMn
2
andFe2inapH-dependentmannerdownaprotongradient
createdbythevacuolarH-ATPase(12–14).Nramp1-medi-
atedexclusionofessentialmetalsmayimpairmicrobialmet-
abolicactivityincludingexpressionofintracellularsurvival
mechanismsormaydirectlyenhancetheefficacyofbacteri-
cidaleffectormechanismsorboth.Nramp2(alsoknownasDMT1andSlc11a2)isaclosehom-
ologofNramp1(15,16)with66%aminoacidsequenceidentity
and77%similaritybetweenthetwoproteins(17).Nramp2is
expressedabundantlyandinaniron-regulatedfashionatthe
brushborderoftheduodenum(18)whereitimportsdietary
ironacrosstheabsorptiveepithelium.Nramp2isalsoexpressed
inmanycelltypesandisabundantinerythroidprecursors(19)
whereitisrequiredforrecruitmentoftransferrin-associated
ironfromrecyclingendosomesintothecytosol(20).Expres-
sionofNramp2hasalsobeendetectedatthebrushborderof
epithelialcellsliningtheproximaltubulesofthekidneywhereit
mayfunctionasareuptakesystemfordivalentmetals(21).Two
majorNramp2proteinisoformsgeneratedbyalternativesplic-
ingat3exonshavebeenidentified.IsoformI(IRE)hasan
iron-responsiveelement(IRE)
2inthe3-untranslatedregion,
whereasisoformII(IRE)lackstheIRE.Inaddition,thecar-
boxyl-terminal18aminoacidsofisoformIarereplacedbyan
alternate25-aminoacidsegmentinisoformII.Nramp2isoform
Iispredominantlyexpressedinepithelialcells,whereasisoform
IIispredominantlyexpressedinerythroidcells.Muchofour
knowledgeofthefunctionofNramp2invivocomesfromstud-
*Thecostsofpublicationofthisarticleweredefrayedinpartbythepayment
ofpagecharges.Thisarticlemustthereforebeherebymarked“advertise-
ment”inaccordancewith18U.S.C.Section1734solelytoindicatethisfact.
□STheon-lineversionofthisarticle(availableathttp://www.jbc.org)containssupplementalTable1andFigs.1–3.
1Towhomcorrespondenceshouldbeaddressed:Dept.ofBiochemistry,McGillUniversity,3655PromenadeSirWilliamOsler,Montreal,Quebec
H3G1Y6,Canada.Tel.:514-398-7291;Fax:514-398-2603;E-mail:philippe.
gros@mcgill.ca.2Theabbreviationsusedare:IRE,iron-responsiveelement;HA,hemaggluti-nin;Ab,antibody;GFP,greenfluorescentprotein;PBS,phosphate-buff-
eredsaline;WT,wild-type;ER,endoplasmicreticulum;NT,amino-terminal;
AP,adaptorprotein.
THEJOURNALOFBIOLOGICALCHEMISTRYVOL.281,NO.42,pp.31677–31688,October20,2006
©2006byTheAmericanSocietyforBiochemistryandMolecularBiology,Inc.PrintedintheU.S.A.
OCTOBER20,2006•VOLUME281•NUMBER42JOURNALOFBIOLOGICALCHEMISTRY31677
byguestonApril3,2018
http://www.jbc.org/
Downloadedfrom
iesofrodentmodelsofmicrocyticanemiaandirondeficiency
includingthemkmouseandtheBelgraderat,thatareboth
causedbythesamemissensemutationinpredictedTM4of
Nramp2(G185R)(22,23).Thisphenotypeisrecapitulatedina
mousemutantwithtargetedinactivationofNramp2
(Nramp2
/)(24).Recentlytwohumanpatientswithsevere
hypochromicmicrocyticanemiaandhepaticironoverload
wereshowntoharbormutationsinNRAMP2(25,26).Inboth
patients,aquantitativereductionintheexpressionoffunc-
tionalNramp2hasbeenidentifiedasthecauseofdisease
(27–29).AlthoughNramp1andNramp2codeforfunctionallyundis-
tinguishablepH-dependentdivalentmetaltransporterswith
similarsubstratespecificities(13),theydiffersharplyintheir
subcellularlocalizations.Nramp1isfoundstrictlyatthelyso-
somalcompartmentofcellswithnoexpressionatthecellsur-
face(8,9).Incontrast,Nramp2isubiquitouslyexpressedatthe
plasmamembraneaswellasinrecyclingendosomes(isoform
II)atsteadystate(18,30).Sequencemotifsfoundinthecyto-
plasmicterminalregionsofmembraneproteinsoftencontrol
theirsubcellulartargetingandtrafficking(31,32).Themost
commonmotifsareeithertyrosine-basedsignalsoftheforms
NPXYorYXX(whereXanyresidue,andlargehydro-
phobicresidues)anddileucine-basedmotifs(LL)(33).Thesub-
cellulartraffickingofNramp1isnotwellunderstood,andspe-
cificcytoplasmicmotifsinvolvedinitslysosomaltargetinghave
notyetbeenidentified.However,wehaveshownpreviously
thatinsertionofahemagglutinin(HA)epitopetaginthefourth
extracytoplasmicloopofNramp1causesthetransportertobe
mistargetedtotheplasmamembranewhereitdisplaysmetal
uptakeactivity(13).Ontheotherhand,traffickingsignalsinthe
aminoandcarboxylcytoplasmicregionsofNramp2havebeen
identifiedandcharacterized.Weandothershaveshownthata
555YLLNT559motifinthecarboxylterminusofNramp2iso-
formIIcontrolstargetingtotransferrin-positiverecycling
endosomes(31,32).Deletionofthismotifortruncationofthe
entirecarboxyl-terminalcytoplasmicsegmentofNramp2iso-
formIIresultsinamutantproteinthatisinternalizedlessrap-
idlyfromtheplasmamembrane,isnotproperlyrecycledback
tothecellsurface,andistargetedtolateendosomesandlyso-
somes(32).Mutagenesisexperimentshavealsoshownthat
motifsintheaminoterminusofNramp2(NPAYandYSCF)
playanadditionalbutlessdeterminantroleintherecycling
endosometargetingofNramp2(32).Inthisstudy,weinvestigatedthesubcellulartargetingand
traffickingpropertiesofNramp1.Forthis,weconstructedchi-
mericproteinsbyexchanginghomologoussegmentsof
Nramp1andNramp2(isoformII).Weexpressedthechimeras
instablytransfectedLLC-PK
1cellsandstudiedtheirexpres-
sion,stability,cellsurfaceexpression,metaltransportactivity,
andsubcellularlocalization.Wedemonstratethatatyrosine-
basedsortingsignaloftheformYXXfunctionsasalysosomal
targetingmotifintheaminoterminusofNramp1.
EXPERIMENTALPROCEDURES
MaterialsandAntibodies—Reagent-gradechemicalswere
purchasedfromSigmaunlessotherwiseindicated.Monoclonal
mouseantibody(Ab)HA.11directedagainsttheinfluenzaHAepitopewaspurchasedfromCovance(Princeton,NJ).Cy3-la-
beledgoatanti-rabbitandhorseradishperoxidase-coupled
donkeyanti-mouseAbswerepurchasedfromJacksonImmu-
noresearchLaboratories(WestGrove,PA).Plasmidsencoding
GFPfusionproteinswerekindgiftsfromDr.D.Williams
(DepartmentofBiochemistry,UniversityofToronto;GFP-syn-
taxin13)andDr.PatriceBoquet(INSERM,France;GFP-
Lamp1).Thegenerationofpolyclonalantibodiesrecognizing
theamino-terminalsegmentsofmurineNramp1andNramp2
proteinshasbeendescribedpreviously(8,30).
PlasmidsandConstructs—Theconstructionofmammalian
expressionplasmidscontainingfull-lengthmurinecDNAsfor
eitherNramp1(N1/pCB6)orNramp2isoformII(N2/pCB6)
weredescribedearlier(34).Full-lengthmurineNramp2cDNA
(N2HA/pCB6)wasmodifiedbythein-frameadditionofan
exofacialHAepitope(YPYDVPDYAS)inthefourthpredicted
extracellularloopasdescribedpreviously(34).Arecombinant
PCRprotocolwasusedtogenerateNramp2-HAchimerasbear-
ingtheamino-(N1N-HA)orcarboxyl-terminal(N1C-HA)seg-
mentsofNramp1.SimilarlyNramp1chimerasbearingtheami-
no-(N2N)orcarboxyl-terminal(N2C)segmentsofNramp2
werecreated.ForN1N-HAandN2Nconstructs,asilentBstBI
restrictionenzymesitewasintroducedintoN2HA/pCB6and
N1/pCB6plasmids(atnucleotidepositions200and155,
respectively)immediatelyprecedingthefirstpredictedtrans-
membranedomainofeachproteinusingprimersN2-BstBIand
N1-BstBI(supplementalTable1).N1N-HA/pCB6andN2N/
pCB6constructswerethencreatedbyexchangingXhoI-BstBI
fragmentsofN2HA/pCB6andN1/pCB6plasmids.N1C-HA
andN2CconstructswerecreatedbyarecombinantPCRampli-
ficationprotocolusingchimericoligonucleotidesN2HA-N1C
andN1-N2C(supplementalTable1)showingcomplementarity
withN1andN2attheboundariesofregionstobeexchanged.
TheN2HA-N1Camplificationproductwasinsertedinto
N2HA/pCB6usingrestrictionenzymesitesSacIandEcoRIto
generateN1C/pCB6.TheN1-N2Camplificationproductwas
insertedintoN1/pCB6usingrestrictionsitesAvrIIandEcoRI
togenerateN2C/pCB6.Alaninesubstitutionmutationsatthe
15YGSI18motifofNramp1werecreatedusingmutagenicprim-
erslistedinsupplementalTable1.Mutantswereintroduced
intoN1N-HA/pCB6usingrestrictionenzymesitesXhoIand
BstBI.TheintegrityofallmutantandchimericcDNAswas
verifiedbyDNAsequencing.CellCulture,Transfection,andImmunoblotting—LLC-PK
1cellswereculturedasdescribedpreviously(32)andtransfected
witheitherN1/pCB6,N2HA/pCB6,orchimericplasmidsusing
Lipofectamine2000(Invitrogen)accordingtoinstructions
fromthemanufacturer.Stablytransfectedcellcloneswere
obtainedafter14daysofselectioninmediumcontainingG418
(1.4mg/ml;Invitrogen),andindividualcoloniesweresubse-
quentlypickedandexpanded.Totalcelllysateswereprepared
andresolvedbySDS-PAGE.Clonesshowingrobustexpression
ofthetransfectedconstructswereidentifiedbyimmunoblot-
tingwitheitheramouseanti-HAAb(forN2HA/pCB6,N1N-
HA/pCB6,andN1C-HA/pCB6),arabbitanti-Nramp2NTAb
(N2N/pCB6),orarabbitanti-Nramp1NTAb(N1/pCB6and
N2C/pCB6)asdescribedpreviously(32).Forexperimentswith
cycloheximide,cellswereincubatedingrowthmediumsupple-
Nramp1LysosomalTargetingMotif
31678JOURNALOFBIOLOGICALCHEMISTRYVOLUME281•NUMBER42•OCTOBER20,2006
byguestonApril3,2018
http://www.jbc.org/
Downloadedfrom
mentedwith20g/mlcycloheximidefortheindicatedtime
intervalspriortolysisandSDS-PAGE.MetalTransportAssay—Measurementofmetaltransportin
transfectedLLC-PK
1cellswascarriedoutusingacalceinfluo-
rescencequenchingassayaswehavedescribedpreviously(34,
35).Calceinacetoxymethylester(MolecularProbes)stock
solutionswerepreparedindimethylsulfoxide,andcellswere
loadedatafinalconcentrationof0.25
Mcalceinacetoxym-
ethylester.Co2solution(2mMcobaltchloride)wasprepared
freshindeionizedwater.Initialratesofmetaltransportwere
calculatedfromtheinitialfluorescencequenchingcurves(34,
35).
CellSurfaceBiotinylation—LLC-PK
1cellmonolayerswere
rinsedtwicewithice-coldphosphate-bufferedsaline(PBS)and
oncewithice-coldboratebuffer(10.0m
Mboricacid,154mM
NaCl,7.2mMKCl,1.8mMCaCl2,pH9.0)andthenincubated
(60minat4°C)inthesamebuffercontainingsulfo-NHS-SS-
biotin(1mg/ml;NHSisN-hydroxysulfosuccinimide;Pierce).
UnreactedbiotinwasremovedbythreewasheswithRPMI
1640medium.Biotinylatedcellswerecollectedandsolubilized
inlysisbuffer(1%TritonX-100,0.2%SDS,50m
MTris-HCl,pH
7.4,150m
MNaCl,20%glycerol)withproteaseinhibitors.
Lysateswerepreclearedbycentrifugation(10,000gfor30
minat4°C),andproteinyieldwasquantifiedbyBradfordassay
(Bio-Rad).Biotinylatedproteins(100
goftotalproteinlysate)
wereisolatedbyovernightincubationat4°Cwith50
lof
ImmunoPureimmobilizedstreptavidinslurry(Pierce)ina
finalvolumeof1mlinlysisbuffer.Streptavidinbeadswere
washedsixtimeswithcoldlysisbuffer,andboundproteins
wereelutedwith2Laemmlibufferatroomtemperature
for30min.ProteinswereseparatedbySDS-PAGEfollowed
byimmunoblotting.QuantificationofCellSurfaceExpressionbyEnzyme-linked
ImmunosorbentAssay—QuantificationofNramp2-HAmole-
culesexpressedatthecellsurfacebyenzyme-linkedimmu-
nosorbentassaywasasdescribedpreviously(32).BrieflyLLC-
PK
1cellsweregrowntoconfluencyin48-wellcultureplates
andfixedwith4%paraformaldehydefor30min.Cellswere
blockedin5%nonfatmilkinPBSfor30min,incubatedwith
anti-HAAb(1:500)for90min,washed,andincubatedwith
secondaryAb(donkeyanti-mouse-horseradishperoxidaseAb,
1:4000)for1h.ForquantificationoftotalNramp2-HAexpres-
sion,cellswerepermeabilizedbyincubationwith0.1%Triton
X-100inPBSfor30minpriortoincubationwithanti-HAAb.
Peroxidaseactivitywasdetectedbyincubatingcellswithhorse-
radishperoxidasesubstrate(0.4mg/mlo-phenylenediamine
dihydrochloride,SigmaFASTOPD)accordingtotheinstruc-
tionsfromthemanufacturer.Reactionswerestoppedafter30
minwith3
MHCl,andabsorbancereadings(492nm)were
takenwithaspectrometer.Foralldeterminations,background
absorbancereadingsfrom(i)nonspecificbindingofsecondary
Aband(ii)nonspecificbindingofprimaryAbtovector-trans-
fectedcellsweresubtractedforeachsample.Cellsurfaceread-
ingswerenormalizedtototalNramp2-HAvaluesforeachcell
cloneandwereexpressedasapercentage.Immunofluorescence—LLC-PK
1cellsstablyexpressing
Nramp1,Nramp2-HA,orchimericproteinsweretransiently
transfectedwithGFPfusionproteinstolabelrecyclingendo-somes(GFP-syntaxin13)orlateendosomesandlysosomes
(GFP-Lamp1).Twenty-fourhourslater,cellswerefixedwith
4%paraformaldehyde(inPBS)for20minandwereblockedand
permeabilizedwithaPBSbuffercontaining0.2%saponinand
5%nonfatmilk(30min).Allantibodiesweredilutedinblocking
solution,andincubationswereperformedfor1hatroomtem-
perature.CellsstablyexpressingN2HA,N2N,andN1C-HA
proteinswerelabeledwithanti-Nramp2NTAb(1:200),
whereascellsexpressingN1,N1N-HA,andN2Cproteinswere
labeledanti-Nramp1NTAb(1:400).Allcellsweresubse-
quentlyincubatedwithagoatanti-rabbitsecondaryAbcoupled
toCy3.CellswerevisualizedusinganAxiovert200Mepifluo-
rescencemicroscopewitha100oilimmersionobjective.Dig-
italimageswereacquiredwithaZeissAxioCamHRmcamera
operatedwithAxioVision4.3.Imageswerecropped,assem-
bled,andlabeledusingAdobePhotoshopandIllustrator
softwares.
RESULTS
ConstructionandExpressionofNramp1andNramp2Chi-
mericProteins—TobetterunderstandthetraffickingofNramp
proteins,wecreatedchimericmoleculesbyexchangingthepre-
dictedcytosolicamino-andcarboxyl-terminalsegmentsof
Nramp1andNramp2.Nramp2chimerascontainingeitherthe
amino(N1N-HA)orcarboxyl(N1C-HA)predictedcytosolic
segmentsofNramp1wereconstructedusingtheNramp2iso-
formII(non-IRE)backbonebearinganexofacialHAtagthat
allowedforrecognitionofcellsurfacemoleculesinintactcells
(Fig.1B).WehaveshownpreviouslythatinsertionofthisHA
taginthefourthpredictedextracytoplasmicloop(EC4)of
Nramp2(Nramp2-HA)doesnotaffectexpression,activity,
subcellularlocalization,orrecyclingpropertiescomparedwith
theunmodifiedprotein(36).Nramp2-HAhasbeenusedexten-
sivelytocharacterizethestructure,function,andsubcellular
traffickingofthisproteininLLC-PK
1cells(27,32,34,36–39).
Nramp1chimerascontainingeithertheamino(N2N)orcar-
boxyl(N2C)predictedcytosolicsegmentsofNramp2isoformII
(non-IRE)wereintroducedintoanunmodifiedNramp1pro-
teinbackbone(Fig.1B).Wehaveshownpreviouslythatan
Nramp1moleculemodifiedbyinsertionofanHAtaginEC4is
mistargetedtotheplasmamembraneoftransfectedChinese
hamsterovarycells(13),precludingtheuseofthatsitefor
epitopetaginsertionforthepresentstudies.AllNramp1and
Nramp2chimeraswereintroducedintoLLC-PK
1porcinekid-
neycells,andcellclonesstablyexpressingindividualvariants
wereselectedandexpandedforanalysis.Fig.2Ashowsatypical
immunoblotofextractspreparedfromcellstransfectedwith
eitherNramp2,Nramp1,orchimericcDNAs.Immunoblots
wereprobedseparatelywithantibodiesagainsttheHAtag,the
aminoterminusofNramp2,andtheaminoterminusof
Nramp1.Wild-type(WT)Nramp2wasdetectedastwoimmu-
noreactivespecies,aminorspeciesat60kDaandamajor
speciesat90–105kDa(Fig.2A).Previousstudiesfromour
grouphaveshownthattheminorspeciescorrespondstothe
precursor“coreglycosylated”protein,whereasthemajorspe-
ciescorrespondstothemature“complexglycosylated”formsof
theprotein(36).InterestinglyN1N-HAwasdetectedastwo
speciesoffasterelectrophoreticmobilitythanWTNramp2,
Nramp1LysosomalTargetingMotif
OCTOBER20,2006•VOLUME281•NUMBER42JOURNALOFBIOLOGICALCHEMISTRY31679
byguestonApril3,2018
http://www.jbc.org/
Downloadedfrom
suggestingasignificantdifferenceintheextentofcoreand
complexglycosylationinthischimericprotein(Fig.2A).We
examinedtheglycosylationstatusofN1N-HAusingtheglyco-
sylationinhibitortunicamycin.Incubationwithtunicamycin
resultedinareductionintheamountofcomplexglycosylated
speciesconcomitantwithanincreaseintheprecursorunglyco-
sylatedspecies(supplementalFig.1).TheN1N-HAprecursor
showedanapparentfasterelectrophoreticmobilitythatthe
precursorforNramp2-HA,suggestingpossibledifferencesin
post-translationalmodificationofthetwoproteinsinLLC-PK
1cellsunrelatedtoglycosylation.Nramp1hasbeenshownpre-
viouslytobehyperphosphorylated(40),anddifferencesinthis
orothertypesofmodificationsmayaccountforthedifferences
inmobility.N1C-HAwasdetectedastwoimmunoreactivespe-
ciesofelectrophoreticmobilitysimilartothatofWTNramp2.However,theproportionoftheimmatureprecursorN1C-HA
protein(60kDa)appearedtobesignificantlygreaterthanthat
foundforWTNramp2,suggestingpossibleincompletematu-
rationofthischimera(Fig.2A).WTNramp1andchimeras
N2NandN2Cweredetectedasimmunoreactivespeciesof
70–75kDa(Fig.2A,
bottompanel).LLC-PK
1cellsexpressing
thedifferentWTandchimericproteinsweretreatedwiththe
translationinhibitorcycloheximidefordifferentperiodsof
time,andthefateofcoreglycosylatedandmatureproteiniso-
formswasanalyzedbyimmunoblotting(Fig.2B).Theseexper-
imentsshowedthatWTNramp2andNramp1aswellasthe
chimerasN1N-HA,N2N,andN2Cquicklymaturedfroma
coreglycosylatedprecursortoafullymatureformthat
remainedstablefora7-hperiod.InterestinglytheN1C-HA
chimeradisplayedincreasedpersistenceofprecursorand
FIGURE1.PredictedtraffickingmotifsanddesignofNramp1/Nramp2chimericproteins.A,sequencesoftheamino-andcarboxyl-terminalregionsof
Nramp1andNramp2areshown,includingthelocationsofpredictedtargetingmotifs(highlightedblack),transmembranedomains1(TM1)and12(TM12),and
thelocationsofthesequenceboundariesforthechimeras(scissors).InNramp1,aminoacidresidues1–54and519–548wereconsideredastheamino-and
carboxyl-terminalregions,respectively.InNramp2,residues1–69and533–568wereconsideredastheamino-andcarboxyl-terminalregions,respectively.B,
Nramp1/2chimeraswerecreatedbyexchangingtheamino-andcarboxyl-terminalsegmentsofNramp1andNramp2.Schematicrepresentationsareshown
ofwild-typeNramp2-HA(gray)andwild-typeNramp1(black)aswellasthefourchimerascreated.Thepositionsofantigenicepitopesrecognizedbytheanti-HA,anti-Nramp1,andanti-Nramp2antibodiesaswellastheputativeN-linkedglycosylationsites(Nramp1:Asn
321andAsn335;Nramp2:Asn336andAsn349)
areindicated.
Nramp1LysosomalTargetingMotif
31680JOURNALOFBIOLOGICALCHEMISTRYVOLUME281•NUMBER42•OCTOBER20,2006
byguestonApril3,2018
http://www.jbc.org/
Downloadedfrom
reducedstabilityofthematureformcomparedwithcontrols
withlittleproteindetectableat7h(Fig.2B).Theseresultssug-
gestimpairedprocessingoftheN1C-HAchimera.CellSurfaceExpressionofChimericProteins—Weusedacell
surfacebiotinylationtechniquetoquantifyplasmamembrane
expressionoftheNramp1/2chimerasatsteadystate(27,32).
LLC-PK
1cellsstablyexpressingeitherWTorchimericNramp
constructsweretreatedwithamembrane-impermeablebiotin
compoundtolabelallcellsurfaceproteins.Aftersolubilization,
cellsurfaceproteinswereisolatedwithimmobilizedstreptavi-
dinfollowedbyseparationbySDS-PAGEandvisualizationby
immunoblotting(Fig.3A,Surface).Cellsurfaceexpressionof
eachproteinwasthencomparedwithtotalproteinexpression
foreachcellline(Fig.3A,Total).WTNramp2-HAand
N1C-HAweredetectedatrobustlevelsattheplasmamem-
brane,whereasWTNramp1,N1N-HA,N2N,andN2Cdis-
playedlittleornocellsurfaceexpression(Fig.3A).Clearly
replacingeithertheamino(N2N)orcarboxyl(N2C)terminiof
Nramp1withtheequivalentsegmentsofNramp2didnotresult
inplasmamembraneexpression.BecauseNramp2-HA,N1N-
HA,andN1C-HAwereconstructedwithexofacialHAtagsin
predictedEC4,thefractionofeachvariantpresentatthecell
surfaceatsteadystatecouldbedeterminedbyexposingfixed
LLC-PK
1transfectantstoanti-HAantibodywith(totalexpres-
sion)orwithout(cellsurface)priorpermeabilizationwith
detergent.Theamountofboundanti-HAantibodywasquan-tifiedusingasecondaryantibodycoupledtohorseradishper-
oxidase.Wedeterminedthat35.9
b3.9%(meanbS.E.)ofWT
Nramp2-HAand21.3b1.3%ofN1C-HAwereexpressedatthe
cellsurfacecomparedwithamodest0.5b1.2%forN1N-HA
(Fig.3B).Thesevaluesareconsistentwithcellsurfacebiotiny-
lationresults(Fig.3A)andsuggestthattheaminoterminusof
Nramp1impairsnormaltargetingofNramp2totheplasma
membrane.Nramp2isoformIIisnormallyexpressedattheplasmamem-
braneandinrecyclingendosomesinLLC-PK
1cells.Replacing
thecarboxylterminusofNramp2-HAwiththeequivalentseg-
mentofNramp1(N1C-HA)didnothaveamajoreffectonthe
cellsurfaceexpressionofthetransporter(Fig.3,AandB).Inter-
estinglydespitetheimpairedprocessingandreducedstability,
N1C-HAretainedsignificantmetaltransportactivity(Fig.3C).
Thisactivitylikelystemsfromthesignificantfractionofmature
N1C-HAproteinthatisexpressedatthecellsurface.Onthe
otherhand,replacingtheaminoterminusofNramp2-HAwith
theequivalentsegmentfromNramp1(N1N-HA)drastically
reducedsurfaceexpression(Fig.3,AandB)aswellasmetal
transportactivity(Fig.3C).Thisresultsuggeststhattheamino
terminusofNramp1containstargetinginformationthataffects
thenormaltraffickingofNramp2totheplasmamembrane.SubcellularLocalizationofChimericProteins—Immunoflu-
orescencewasusedtocomparethesubcellularlocalizationof
wild-typeandchimericproteinsatsteadystate.LLC-PK
1trans-
FIGURE2.
ExpressionandstabilityofNramp1/2chimerasintransfectedLLC-PK1cells.Totalcellextractsfromuntransfectedcells(LLC-PK1)andcellclones
stablyexpressingwild-typeNramp1(Nramp1),wild-typeNramp2(Nramp2-HA),andtheNramp1/2chimeras(N1N-HA,N1C-HA,N2N,andN2C)wereresolvedby
SDS-PAGEandanalyzedbyimmunoblotting.A,immunoblotswereincubatedwithantibodiesrecognizingeithertheHAepitope(anti-HA),theaminoterminus
ofNramp2(anti-Nramp2),ortheaminoterminusofNramp1(anti-Nramp1).Thesizesofmolecularmassstandards(inkilodaltons)areindicated.InB,transfec-
tantsexpressingtheindicatedproteinsweretreatedwiththetranslationinhibitorcycloheximide(CHX;20
g/ml)for0,1,3,5,or7h.Immunoblottingoftotal
celllysateswascarriedouttoassessthestabilityofwild-typeandchimericproteins.Antibodiesusedforeachblotareindicated.
Nramp1LysosomalTargetingMotif
OCTOBER20,2006•VOLUME281•NUMBER42JOURNALOFBIOLOGICALCHEMISTRY31681
byguestonApril3,2018
http://www.jbc.org/
Downloadedfrom
fectantswerefixedandpermeabilized,andchimericproteins
werelabeledwithpolyclonalantibodiesrecognizingeither
Nramp1orNramp2.Consistentwithourpreviouslypublished
data(32,36),Nramp2-HAdisplayedstrongcolocalizationwith
GFP-syntaxin13,whereasNramp1showedlittleoverlapwith
therecyclingendosomemarker(Fig.4A).Interestinglyallchi-
merasshowednosignificantcolocalizationwithGFP-syntaxin
13(Fig.4A).Theseresultssuggestthatsubstitutionofeitherthe
aminoorcarboxylterminusofNramp2byhomologousseg-
mentsofNramp1impairstargetingtotherecyclingendosome
compartment.Consistentwithpreviouslypublisheddata(8),Nramp1
showedstrongcolocalizationwiththelysosomalmarkerGFP-
Lamp1,whereasNramp2-HAdisplayedlittlecolocalization
withthismarker(Fig.4B).TheseresultsconfirmthatNramp1
isproperlytargetedtothelysosomalcompartmentinLLC-PK
1cells.Remarkablyallchimerasdisplayedsignificantcolocaliza-
tionwithGFP-Lamp1(Fig.4B),indicatingtheirpresenceinlate
endosomesandlysosomes.Clearlyreplacingtheaminoorcar-
boxylterminiofNramp1withtheequivalentregionsof
Nramp2(N2NandN2C)didnotsignificantlyaffectthesubcel-lulardistributionofNramp1at
steadystate(Table1).Previous
workbyusandothershasdemon-
stratedthatthecarboxylterminus
ofNramp2isoformIIcontainstar-
getinginformation(includinga
YLLNTsignal)crucialforendocy-
tosisandrecyclingofthetrans-
porterfromtheplasmamembrane
(31,32).Mutationsordeletionsin
thismotifresultinaproteinthathas
impairedrecyclingfollowinginter-
nalizationfromthecellsurfaceand
accumulatesinthelysosomalcom-
partment(32).Consistentwith
thesefindings,replacingthecar-
boxylterminusofNramp2withthe
equivalentsegmentfromNramp1
(N1C-HA)resultedinplasmamem-
braneexpression(Fig.3)andlysoso-
maltargeting(Fig.4B
andTable1).
Therefore,themistargetingdis-
playedbyN1C-HAisprobablydue
toremovalofthecarboxylterminus
ofNramp2ratherthanthepresence
ofadominanttargetingsignalinthe
carboxylterminusofNramp1.A
fractionofN1C-HAshowedsome
colocalizationwiththeendoplasmic
reticulummarkerGFP-Sec61(data
notshown),suggestingthatsome
N1C-HAmayberetainedintheER.
Finallysubstitutingtheaminoter-
minusofNramp2withtheequiva-
lentsegmentfromNramp1(N1N-
HA)yieldedthemostintriguing
result.N1N-HAshowedsubcellular
localizationindistinguishablefromWTNramp1withnosignif-
icantsurfaceexpression(Table1)butstronglysosomaltarget-
ing(Fig.4B).Theseresultssuggestthattheaminoterminusof
Nramp1possesseslysosomaltargetinginformationthatcanact
inadominantfashionovertherecyclingendosomesignalpres-
entinthecarboxylterminusofNramp2isoformII.CharacterizationoftheYSGIMotifintheAminoTerminusof
Nramp1—Closerexaminationofthepredictedcytoplasmic
amino-terminalregionofNramp1revealeda
15YGSI18motif
thatfitstheYXXconsensussignature(Fig.1A).YXXmotifs
havebeenimplicatedintargetingmembraneproteinssuchas
LAMP-1andLAMP-2tolysosomesbyinteractingwiththe
subunitsofAP-1andAP-2complexes(33,41–44).Todeter-
minewhethertheYGSImotifintheaminoterminusofNramp1
wasresponsibleforthelysosomaltargetingoftheN1N-HA
chimera,wecreatedalaninesubstitutionsateachpositionof
theYGSIsignalwithintheN1N-HAchimericconstruct.The
mutantswerestablytransfectedintoLLC-PK
1cells,andclones
positiveforexpressionwereselectedforanalysis.Immunoblot
analysisperformedoncellextractsshowedstableexpressionof
Y15A,G16A,andS17AmutantsatlevelscomparabletoN1N-
FIGURE3.CellsurfaceexpressionandmetaltransportactivityoftheNramp1/2chimeras.A,cellsurface
biotinylationwasusedtoassessplasmamembraneexpressionofwild-typeNramp1(Nramp1),wild-type
Nramp2(Nramp2-HA),andtheNramp1/2chimeras(N1N-HA,N1C-HA,N2N,andN2C).Livecellswerelabeled
withamembrane-impermeablebiotincompound(see“ExperimentalProcedures”).Totalcellproteinextracts
wereprepared,andbiotinylatedproteinswereisolatedbyaffinitycapturewithstreptavidin-agarosebeads.
Equivalentamountsoftotalcellextracts(Total)andcapturedbiotinylatedprotein(Surface)wereresolvedbySDS-PAGEfollowedbyimmunoblottingwithanti-Nramp2(i)oranti-Nramp1(ii)polyclonalantibodies.B,the
fractionofNramp2-HA,N1N-HA,andN1C-HAexpressedatthecellsurfacewasquantifiedusingtheexofacial
HAepitopetaginsertedinpredictedextracytoplasmicloop4ofthoseproteins.Brieflycellswerefixedand
incubatedwithprimaryanti-HAantibodywithorwithoutpriordetergentpermeabilizationfollowedbyincu-
bationwithahorseradishperoxidase-coupledsecondaryantibodyandquantificationbyacolorimetricassay.
TheamountofNrampproteinsexpressedatthecellsurface(innon-permeabilizedcells)isshownasafraction
(%)oftotalproteinexpression(inpermeabilizedcells).InC,metaltransportactivityoftheNramp1/2chimeras
wasdeterminedusingafluorescencequenchingassay.Cellsloadedwithametal-sensitivefluorescentdye
wereincubatedwithCo
2inacidicbuffer.Resultsareshownastheinitialratesoffluorescencequenching.
ErrorbarsrepresentS.E.ofthreeormoreindependentdeterminations.
Nramp1LysosomalTargetingMotif
31682JOURNALOFBIOLOGICALCHEMISTRYVOLUME281•NUMBER42•OCTOBER20,2006
byguestonApril3,2018
http://www.jbc.org/
Downloadedfrom
HA,althoughlowerexpressionwasdetectedforI18A(Fig.5A).
WedeterminedthefractionofeachN1N-HAexpressedatthe
cellsurface.MutatinganyoftheresiduesintheYGSImotiftoalanineincreasedthesurfaceexpressionofthechimera(Fig.
5B).Y15Ademonstratedthehighestsurfaceexpression(19.2
b
2.0%)followedbyS17A(10.2b1.2%),I18A(7.4b2.1%),and
G16A(4.4b0.3%).Thisincreaseinsurfaceexpressionwas
concomitanttoacommensurateincreaseinmetaltransport
activityforthemutants(Fig.5C),implyingthatN1N-HAis
indeedproperlyfoldedinatransport-competentmanneratthe
cellsurface.WedeterminedthesubcellularlocalizationoftheN1N-HA
mutantsatsteadystateinfixedandpermeabilizedcells.
MutantsG16AandS17Adisplayedsubcellularlocalization
similartoN1N-HA,showinglittlecolocalizationwiththerecy-
FIGURE4.SubcellularlocalizationofNramp1/2chimeras.LLC-PK1cellsstablyexpressingwild-typeNramp1(Nramp1),wild-typeNramp2(Nramp2-HA),and
theNramp1/2chimeras(N1N-HA,N1C-HA,N2N,andN2C)weretransientlytransfectedwitheitherGFP-syntaxin13tolabelrecyclingendosomes(A)or
GFP-Lamp1tolabellateendosomesandlysosomes(B).Twenty-fourhourslater,cellswerefixed,permeabilized,andstainedwitheitheranti-Nramp1or
anti-Nramp2polyclonalantibodiesfollowedbyasecondaryantibodycoupledtofluorescentCy3.Imageswereacquiredbyepifluorescencemicroscopy.Insets
showmagnificationsoftheareaboxedinthefigure.
TABLE1
SubcellularlocalizationofNramp1/2chimeras
Plasma
membraneRecycling
endosomesLateendosomes
andlysosomesEndoplasmic
reticulum
Nramp2-HA
N1N-HA
N1C-HA
Nramp1
N2N
N2C
Nramp1LysosomalTargetingMotif
OCTOBER20,2006•VOLUME281•NUMBER42JOURNALOFBIOLOGICALCHEMISTRY31683
byguestonApril3,2018
http://www.jbc.org/
Downloadedfrom
clingendosomemarkerGFP-syntaxin13(Fig.6A)butstrong
colocalizationwiththelysosomalmarkerGFP-Lamp1(Fig.6B).
TheseresultssuggestthatGly
16andSer17arenotessentialfor
thelysosomaltargetingofN1N-HA.StrikinglymutantsY15A
andI18AdisplayedsubcellularlocalizationsimilartoWT
Nramp2,showingstrongcolocalizationwithsyntaxin13-posi-
tiverecyclingendosomes(Fig.6A)butlittlecolocalizationwith
Lamp1-positivelysosomes(Fig.6BandsummarizedinTable
2).TheseresultssuggestthatTyr
15andIle18arecriticalforthe
lysosomaltargetingofN1N-HAandthattheYGSImotifinthe
aminoterminusofNramp1functionsasaYXXlysosomal
targetingsignal.FurthermorethisYGSIsignalappearstodom-
inatetherecyclingendosomesignalpresentinthecarboxyl
terminusofNramp2isoformII.
DISCUSSION
Inthisstudy,wesoughttoinvestigatethesubcellulartargeting
andtraffickingpropertiesofNramp1,includingtheidentification
ofsortingsignalsresponsibleforitsdistinctlocalizationinlyso-
somesofmononuclearphagocytes.Forthis,weconstructedchi-
mericproteinsinwhichhomologousdomainsofNramp1andthe
closelyrelatedirontransporterNramp2(isoformII)were
exchanged.Replacingtheamino(N2N)orcarboxyl(N2C)termi-nalcytoplasmicsegmentsofNramp1
withtheequivalentsegmentsfrom
Nramp2didnotdrasticallyaffectthe
expressionorsubcellularlocalization
ofthetransporter.IndeedbothN2N
andN2Cchimerasexhibitedlittlecell
surfaceexpressionbutshowedstrong
lysosomaltargetingtypicalofWT
Nramp1.Theseresultssuggestedthat
sortingsignalsintheaminoandcar-
boxylcytoplasmicterminiofNramp2
areunabletoconfercellsurface
and/orrecyclingendosometargeting
totheNramp1backbone.Results
withtheN2Nchimeraaresupported
byourpreviousworkshowingthat
deletionmutationsatputativesorting
signalsintheaminoterminusof
Nramp2donotsignificantlyaffectthe
cellsurfaceorrecyclingendosome
targetingofthetransporter(32).In
contrast,weandothergroupshave
shownthataYLLNTmotifinthecar-
boxylterminusofNramp2isoformII
controlstheinternalizationand
recy-
clingoftheproteinfromplasmamem-
brane(31,32).However,resultswith
theN2Cchimerasuggestedthatthis
YLLNTmotifwassomehowmaskedor
non-functionalwhenplacedonthe
backgroundofNramp1.AnNramp2-HAchimerabearing
thecarboxyl-terminalcytoplasmic
segmentofNramp1(N1C-HA)was
robustlyexpressedinLLC-PK
1cells
butdisplayedahigherfractionofa60-kDaprecursorspecies
andalowerfractionofthe90-kDamatureprotein,suggesting
impairedprocessingofthischimera.N1C-HAalsodisplayed
reducedstability.Surfacebiotinylationexperimentsrevealed
thatthemature,fullyglycosylatedN1C-HAwasexpressedat
thecellsurface(Fig.2A)andwaslikelyresponsiblefortheresid-
ualmetaltransportactivityobservedforthisconstruct(Fig.
2C).Thisresultsuggeststhatcomplexglycosylationacquired
bytheprecursorproteinintheGolgiapparatusiscriticalforthe
plasmamembranetargetingofmatureNramp2.However,a
morelikelyexplanationisthatprocessingoftheoligosaccha-
ridesismerelyareflectionthattheproteinswerefoldedwell
enoughtoexittheERandthereafteracquirecomplexglycosy-
lation.Strikinglythemultiplebiosyntheticandfunctional
defectsoftheN1C-HAchimeravirtuallymirrorthoseobserved
fortheNramp2
G185Rmutantproteinproducedinmkmiceand
Belgraderats(37).AlthoughasmallfractionofN1C-HAdis-
playedsomecolocalizationwiththeERmarkerGFP-Sec61
(datanotshown),amoresignificantfractionofthechimerawas
expressedinlateendosomesandlysosomes.Wehaveshown
previouslythatcriticalmutationsatthecarboxylterminusof
Nramp2isoformII(includingatruncationoftheentirecarbox-
yl-terminalsegment)resultinaproteinthatisunabletorecycle
FIGURE5.CharacterizationofN1N-HAtargetingmotifmutants.A,totalcellextractswerepreparedfrom
transfectantsstablyexpressingindividualmutants(Y15A,G16A,S17A,andI18A)intheputativeYGSIlysosomal
targetingmotifpresentintheN1N-HAbackbone.EqualamountsofcellextractwereresolvedbySDS-PAGE
andanalyzedbyimmunoblottingwithanti-HAantibody.Thesizesofmolecularmassstandards(inkilodaltons)
areindicated.InB,thefractionofN1N-HAexpressedatthecellsurfacewasdeterminedasdescribedinthe
legendofFig.3B.C,metaltransportactivityoftheN1N-HAtargetingmotifmutantswasmeasuredbyquench-
ingofcalceinfluorescenceasdescribedinthelegendofFig.3C.ErrorbarsrepresentS.E.ofthreeormore
independentdeterminations.
Nramp1LysosomalTargetingMotif
31684JOURNALOFBIOLOGICALCHEMISTRYVOLUME281•NUMBER42•OCTOBER20,2006
byguestonApril3,2018
http://www.jbc.org/
Downloadedfrom
afterinternalizationfromthecellsurfaceandistargetedtothe
lysosomesbydefault(32).Therefore,wereasonedthatthelyso-
somaltargetingdisplayedbyN1C-HAlikelyresultsfromthe
removalofthecarboxylterminusofNramp2ratherthanthe
presenceofadominanttargetingsignalinthecarboxyltermi-
nusofNramp1.AnNramp2chimerabearingtheaminoterminusofNramp1
(N1N-HA)yieldedperhapsthemostintriguingresults.
N1N-HAwasstablyexpressedintransfectedLLC-PK
1cellsasa
proteinoflowerapparentmolecularweightcomparedwith
Nramp2-HA(Fig.2A)probablyduetoalteredglycosylationof
thechimera.Interestinglytheextentofglycosylationof
N1N-HAresembledmorecloselythatofNramp1(Fig.2A)and
raisesthepossibilitythattheaminoterminusofNramp2con-
tainssequenceinformationthataffectstheextentofglycosyla-
tionofthetransportereitherdirectlyorindirectlybymediating
itstraffickingandretentiontospecificsubcellularcompart-
ments.StrikinglyN1N-HAalsodisplayedsubcellulartargetingpropertiessimilartoNramp1withnosurfaceexpressionyet
stronglysosomaltargeting(Figs.3and4).Theseresults
stronglysuggestedthattheamino-terminalcytoplasmicseg-
mentofNramp1containedlysosomaltargetinginformation
thatwasabletoactinadominantfashionoveraknownrecy-
clingendosometargetingsignal(
555YLLNT559)inthecarboxyl
terminusofNramp2isoformIIpresentinN1N-HA.Thelackof
significanttransportactivityforN1N-HA,asforNramp1,likely
resultedfromthelackofexpressionatthesiteoftransport
measurements(plasmamembrane)ratherthanexpressionofa
non-functionalprotein(Fig.3).Closerexaminationofthe
aminoacidsequencewithintheaminoterminusofNramp1
revealedapotentialtyrosine-basedsignaloftheformYXX
(
15YGSI18)(Fig.1A).AlaninesubstitutionmutationsatTyr15
andIle18withintheN1N-HAchimerawereabletorestorecell
surfaceandrecyclingendosometargetingoftheNramp2chi-
mera(Figs.5Band6B).However,alaninemutationsatGly
16
andSer17ofN1N-HAretainedtheirlysosomaltargeting(Fig.
6B).Theseresultsclearlydemonstratethat15YGSI18functions
asalysosomaltargetingmotifthatfitstheconsensusYXXin
theaminoterminusofNramp1.YXXsignalshavebeenshown
tofunctionaslysosomalsortingmotifsinmembraneproteins
suchasLAMP-1(YQTI)(45).YXXsignalsareknowntobind
the
subunitsofAPcomplexesstrictlythroughtheirYand
residues;however,theXresiduesandotherresiduesflanking
themotifcontributetothestrengthandspecificityofthesig-
FIGURE6.SubcellularlocalizationofN1N-HAmutants.LLC-PK1cellsstablyexpressingN1N-HAYGSImutantsweretransientlytransfectedwitheitherthe
recyclingendosomemarkerGFP-syntaxin13(A)orthelateendosomalandlysosomalmarkerGFP-Lamp1(B).Twenty-fourhourslater,cellswerefixed,
permeabilized,andstainedwithananti-Nramp1polyclonalantibodyfollowedbyasecondaryantibodycoupledtofluorescentCy3.Imageswereacquiredby
epifluorescencemicroscopy.Insetsshowmagnificationsoftheareaboxedinthefigure.
TABLE2
SubcellularlocalizationofN1N-HAmutants
Plasma
membraneRecycling
endosomesLateendosomes
andlysosomesEndoplasmic
reticulum
N1N-HA
Y15A
G16A
S17A
I18A
Nramp1LysosomalTargetingMotif
OCTOBER20,2006•VOLUME281•NUMBER42JOURNALOFBIOLOGICALCHEMISTRY31685
byguestonApril3,2018
http://www.jbc.org/
Downloadedfrom
nals(33).Althoughthe15YGSI18motifintheaminoterminusof
Nramp1canclearlyfunctionasalysosomaltargetingsignal,
otherdeterminantswithinNramp1alsoappeartobeimportant
forlysosomaltargetingoftheprotein.Thisissupportedbytwo
observations.First,theN2Nchimera,whichbearstheamino
terminusofNramp2onthebackboneofNramp1,wasableto
retainitslysosomaltargetingdespitelackingtheYGSImotif.
However,wecannotexcludethepossibilitythatthe
62YSCF65
motifpresentintheaminoterminusofNramp2mayfunction
asalysosomaltargetingsignalinN2Nthatisnormallymasked
bythepresenceoftherecyclingendosometargetingmotifin
thecarboxylterminusofNramp2isoformII(
555YLLNT559).
Second,YGSIsignalmutantsintroducedintoWTNramp1
seemedtoretaintheirlysosomallocalization(supplemental
Fig.3,AandB).However,wenotedthatsomeoftheNramp1
YGSImutantsdisplayedanincreasedamountofplasmamem-
braneexpressionmeasuredbycellsurfacebiotinylationthatis
notnormallyseenforWTNramp1(supplementaryFig.2A).
Thisincreaseincellsurfaceexpressionwasconcomitantwith
anincreaseinmetaltransportactivityattheplasmamembrane
(supplementalFig.2B).Theincreasedcellsurfaceexpressionof
certainNramp1YGSImutantssuggeststhattheplasmamem-
branemaybeadefaultpathwayforNramp1thatmaybe
positivelymodulatedbytheYGSImotiftodirectlysosomal
targeting.
TheidentificationofanNramp1lysosomaltargetingsig-
nalallowsustoproposeamodelfortheroleofcytoplasmic
motifsinsubcellulartraffickingofNramp1andNramp2.In
thismodel(Fig.7),Nramp2isoformIIissynthesizedinthe
ER,targetedtotheGolgiapparatusforcomplexglycosyla-
tion,anddispatchedtotheplasmamembrane.Atthecell
surface,Nramp2recruitsspecificadaptorproteinsrequired
forrapidclathrin-mediatedendocytosisthroughbindingto
aYLLNTmotifpresentinitscarboxylterminus.Nramp2is
theninternalizedintoearlyendosomes,andtherecruited
adaptorcomplexesareinvolvedinsignalingtherecyclingof
thetransporterbacktothecellsurfaceviarecyclingendo-
somes(Fig.7).Thispathwayissharedbythetransferrin
receptorandisofcriticalimportanceforironacquisition
fromtransferrinfollowingacidificationoftheendosomes
andNramp2-mediatedtranslocationofirontothecyto-
plasm.SimilartoNramp2,Nramp1istargetedfromtheER
totheGolgiforcomplexglycosylation.However,therecog-
nitionoftheYGSIsignalbyspecificadaptorproteinsmedi-
atesthesortingofNramp1directlyfromthetrans-Golgi
networktolateendosomesandlysosomes(Fig.7).The
N1N-HAchimera,whichpossessestheNramp1YGSIsignal,
istargetedinafashionsimilartoWTNramp1withtheami-
no-terminallysosomalsignaltakingprecedenceoverthe
carboxyl-terminalYLLNTrecyclingsignal.N1N-HA
Y15A
andN1N-HAI18Amutantsareunabletorecruitlysosomal
sortingproteinsandaretargetedtorecyclingendosomesvia
thecarboxyl-terminalYLLNTsignalinafashionsimilarto
WTNramp2(Fig.7).Ontheotherhand,althoughalanine
mutationsatGly
16andSer17ofN1N-HAdonotdrastically
affectlysosomaltargeting;themutationsmayreducethe
effectivenessoftheYXXsignal.Thisreducedsortingeffi-
ciencymayresultinahigherfractionofN1N-HA
G16AandN1N-HA
S17Amutantsbeingsortedtothecellsurfaceand
subsequentlytargetedtothelysosomalcompartmentviathe
endocyticpathway(Fig.7).Ithasbeenshownthatmutations
intheXresiduesorinotherresiduesneighboringtheYXX
signalcanimpairlysosomaltargetingwithoutsignificantly
disruptinginternalization(43,46,47).Anindirecttargeting
ofN1N-HA
G16AandN1N-HAS17Amutantstolysosomesvia
theplasmamembranewouldexplaintheincreasedcellsur-
faceexpressionobservedforthesemutants(Fig.5B).
AnotherpossibilityisthatWTNramp1normallytrafficsto
thelysosomeviarapidinternalizationfromtheplasmamem-
brane.However,alargeamountofpublisheddataindicates
thatthereislittleifanyNramp1expressionatthecellsurface
atsteadystate(8,13,34),suggestingthatanytrafficking
throughtheplasmamembranemustbeaminorcomponent
oftheoveralllysosomaltargetingpathwayforNramp1.
FIGURE7.ModelforsubcellulartraffickingofNramp1andNramp2(iso-
formII).Nramp2isoformIIissynthesizedintheER,targetedtotheGolgi
apparatus(step1)forcomplexglycosylation,andtargetedtotheplasma
membrane(step2).Atthecellsurface,Nramp2isinternalizedviaaclathrin-
anddynamin-dependentprocessbyrecruitingadaptorproteinsthatspecif-
icallyrecognizethecarboxyl-terminalYLLNTmotif(step3).Nramp2isthentargetedtoearlyendosomes,andtheinteractionofYLLNTwithadaptorpro-
teinsmediatesitsrecyclingbacktothecellsurfaceviarecyclingendosomes
(steps4and5).Nramp1issimilarlytraffickedtotheGolgiaftersynthesis(step
1).However,recognitionoftheYGSIsignalbylysosomalsortingproteins
mediatesitstargetingfromthetrans-Golginetworkdirectlytolateendo-
somesandlysosomes(steps7and8).TheNramp2-HAchimeraN1N-HA,
whichpossessestheNramp1amino-terminalYGSIsignal,istargetedina
fashionsimilartoWTNramp1.N1N-HA
Y15AandN1N-HAI18Amutantsare
unabletorecruitlysosomalsortingproteinsandaretargetedtorecycling
endosomesviathecarboxyl-terminalYLLNTsignalinafashionsimilartoWT
Nramp2.N1N-HA
G16AandN1N-HAS17Amutantsaresortedlessefficientlyto
lysosomes,leadingtosomecellsurfaceaccumulation.Thesemutants,how-
ever,arenotrecycleduponinternalizationandareultimatelytargetedto
lysosomesviathenormalendocyticpathway.RE,recyclingendosomes;LE,
lateendosomes.
Nramp1LysosomalTargetingMotif
31686JOURNALOFBIOLOGICALCHEMISTRYVOLUME281•NUMBER42•OCTOBER20,2006
byguestonApril3,2018
http://www.jbc.org/
Downloadedfrom
InterestinglytheYGSIsignalinNramp1doesnotcompletely
conformtotheYXXsignalsfoundinseveralotherlysosomal
proteins,raisingthepossibilitythatYGSImayrepresentanovel
subsetofYXXsignals.TwocommonfeaturesofYXXsig-
nalsinvolvedinlysosomaltargetingseemtobethepresenceof
aglycineresidueprecedingthecriticaltyrosineaswellasthe
prevalenceofthemotifsinthecarboxylterminusofmem-
braneproteins(33).Nramp1possessesaserineinsteadofa
glycineupstreamofitsYGSIsignal.AlsotheYGSIsignalis
foundintheaminoterminusoftheproteinasopposedtothe
morecommonlyfoundcarboxylterminus.Amino-terminal
YXXmotifshavebeenidentifiedinproteinssuchasthetrans-
ferrinreceptor(YTRF)butfunctiontypicallyasendocyticsig-
nals.Additionalexperimentsareneededtoidentifythespecific
bindingpartnersofYGSIandbetterunderstandthemolecular
machineryinvolvedinthesortingofNramp1tolateendosomes
andlysosomes.Hopefullythecharacterizationofothertyro-
sine-basedsignalsinthecytoplasmicdomainsoflysosomal
proteinswillclarifywhethertheYGSImotifinNramp1repre-
sentsanovelsubsetofYXXmotifs.
ClearlyproperlysosomaltargetingofNramp1iscriticalfor
itsantimicrobialactivityasametaleffluxpumpatthephago-
somalmembraneofmacrophagesandotherphagocyticcells.
Uponphagocytosisofalivepathogen,Nramp1-positivelyso-
somesfusewiththemembranesofthepathogen-containing
phagosomes,andNramp1remainsassociatedwiththepha-
gosomesthroughtheirmaturationtofullyantimicrobial
phagolysosomes(8).Ourdiscoverythatasequencemotifin
theaminoterminusofNramp1issufficienttotargetNramp
proteinstothelysosomessuggestsamodelforfunctional
evolutionofNrampproteins.Inthismodel,Nramp1and
Nramp2weregeneratedbygeneduplicationfromacommon
ancestorfollowedbysubsequentsequencedivergenceto
retainfunctionwithrespecttomechanismoftransportand
ionselectivity.Furtherdivergence,including(a)restriction
ofNramp1transcriptionalactivitytocellsofthemononu-
clearandpolymorphonuclearphagocytelineagesand(b)
acquisitionofatargetingsignalintheaminoterminus
(YGSI)activelydirectingNramp1localizationtolysosomes
fordeliverytopathogen-containingphagosomes,would
haveensuedtoenablemetaleffluxatthephagosomalmem-
brane.Thisactivityappearstohavebeenextremelybenefi-
cialtothehostandhasbeenpreservedthroughoutevolution
fromlowereukaryotestohumans(17).
REFERENCES
1.Forbes,J.R.,andGros,P.(2001)TrendsMicrobiol.9,397–403
2.Vidal,S.M.,Malo,D.,Vogan,K.,Skamene,E.,andGros,P.(1993)Cell73,
469–485
3.Malo,D.,Vogan,K.,Vidal,S.,Hu,J.,Cellier,M.,Schurr,E.,Fuks,A.,Bumstead,N.,Morgan,K.,andGros,P.(1994)Genomics23,51–61
4.Vidal,S.,Gros,P.,andSkamene,E.(1995)J.Leukoc.Biol.58,382–390
5.Abel,L.,Sanchez,F.O.,Oberti,J.,Thuc,N.V.,Hoa,L.V.,Lap,V.D.,Skamene,E.,Lagrange,P.H.,andSchurr,E.(1998)J.Infect.Dis.177,
133–145
6.Bellamy,R.,Ruwende,C.,Corrah,T.,McAdam,K.P.,Whittle,H.C.,andHill,A.V.(1998)N.Engl.J.Med.338,640–644
7.Awomoyi,A.A.,Marchant,A.,Howson,J.M.,McAdam,K.P.,Blackwell,J.M.,andNewport,M.J.(2002)J.Infect.Dis.186,1808–1814
8.Gruenheid,S.,Pinner,E.,Desjardins,M.,andGros,P.(1997)J.Exp.Med.185,
717–730
9.Searle,S.,Bright,N.A.,Roach,T.I.,Atkinson,P.G.,Barton,C.H.,Meloen,R.H.,andBlackwell,J.M.(1998)J.CellSci.111,2855–2866
10.Govoni,G.,Canonne-Hergaux,F.,Pfeifer,C.G.,Marcus,S.L.,Mills,S.D.,Hackam,D.J.,Grinstein,S.,Malo,D.,Finlay,B.B.,andGros,P.(1999)
Infect.Immun.67,2225–2232
11.Cuellar-Mata,P.,Jabado,N.,Liu,J.,Furuya,W.,Finlay,B.B.,Gros,P.,andGrinstein,S.(2002)J.Biol.Chem.277,2258–2265
12.Jabado,N.,Jankowski,A.,Dougaparsad,S.,Picard,V.,Grinstein,S.,andGros,P.(2000)J.Exp.Med.192,1237–1248
13.Forbes,J.R.,andGros,P.(2003)Blood102,1884–1892
14.Jabado,N.,Cuellar-Mata,P.,Grinstein,S.,andGros,P.(2003)Proc.Natl.
Acad.Sci.U.S.A.100,6127–6132
15.Gruenheid,S.,Cellier,M.,Vidal,S.,andGros,P.(1995)Genomics25,
514–525
16.Gunshin,H.,Mackenzie,B.,Berger,U.V.,Gunshin,Y.,Romero,M.F.,Boron,W.F.,Nussberger,S.,Gollan,J.L.,andHediger,M.A.(1997)
Nature388,482–488
17.Cellier,M.,Prive,G.,Belouchi,A.,Kwan,T.,Rodrigues,V.,Chia,W.,andGros,P.(1995)Proc.Natl.Acad.Sci.U.S.A.92,10089–10093
18.Canonne-Hergaux,F.,Gruenheid,S.,Ponka,P.,andGros,P.(1999)Blood
93,4406–4417
19.Canonne-Hergaux,F.,Zhang,A.S.,Ponka,P.,andGros,P.(2001)Blood
98,3823–3830
20.Hentze,M.W.,Muckenthaler,M.U.,andAndrews,N.C.(2004)Cell117,
285–297
21.Canonne-Hergaux,F.,andGros,P.(2002)KidneyInt.62,147–156
22.Fleming,M.D.,Trenor,C.C.,III,Su,M.A.,Foernzler,D.,Beier,D.R.,Dietrich,W.F.,andAndrews,N.C.(1997)Nat.Genet.16,383–386
23.Fleming,M.D.,Romano,M.A.,Su,M.A.,Garrick,L.M.,Garrick,M.D.,andAndrews,N.C.(1998)Proc.Natl.Acad.Sci.U.S.A.95,
1148–1153
24.Gunshin,H.,Fujiwara,Y.,Custodio,A.O.,Direnzo,C.,Robine,S.,andAndrews,N.C.(2005)J.Clin.Investig.115,1258–1266
25.Mims,M.P.,Guan,Y.,Pospisilova,D.,Priwitzerova,M.,Indrak,K.,Ponka,P.,Divoky,V.,andPrchal,J.T.(2005)Blood105,1337–1342
26.Iolascon,A.,d’Apolito,M.,Servedio,V.,Cimmino,F.,Piga,A.,andCamaschella,C.(2006)Blood107,349–354
27.Lam-Yuk-Tseung,S.,Mathieu,M.,andGros,P.(2005)BloodCellsMol.
Dis.35,212–216
28.Priwitzerova,M.,Nie,G.,Sheftel,A.D.,Pospisilova,D.,Divoky,V.,andPonka,P.(2005)Blood106,3985–3987
29.Gunshin,H.,Jin,J.,Fujiwara,Y.,Andrews,N.C.,Mims,M.,andPrchal,J.(2005)Blood106,2221–2222
30.Gruenheid,S.,Canonne-Hergaux,F.,Gauthier,S.,Hackam,D.J.,Grinstein,S.,andGros,P.(1999)J.Exp.Med.189,831–841
31.Tabuchi,M.,Tanaka,N.,Nishida-Kitayama,J.,Ohno,H.,andKishi,F.(2002)Mol.Biol.Cell13,4371–4387
32.Lam-Yuk-Tseung,S.,Touret,N.,Grinstein,S.,andGros,P.(2005)Bio-
chemistry44,12149–12159
33.Bonifacino,J.S.,andTraub,L.M.(2003)Annu.Rev.Biochem.72,
395–447
34.Picard,V.,Govoni,G.,Jabado,N.,andGros,P.(2000)J.Biol.Chem.275,
35738–35745
35.Lam-Yuk-Tseung,S.,Govoni,G.,Forbes,J.,andGros,P.(2003)Blood101,
3699–3707
36.Touret,N.,Furuya,W.,Forbes,J.,Gros,P.,andGrinstein,S.(2003)J.Biol.
Chem.278,25548–25557
37.Touret,N.,Martin-Orozco,N.,Paroutis,P.,Furuya,W.,Lam-Yuk-Tseung,S.,Forbes,J.,Gros,P.,andGrinstein,S.(2004)Blood104,
1526–1533
38.Lam-Yuk-Tseung,S.,andGros,P.(2006)Biochemistry45,2294–2301
39.Lam-Yuk-Tseung,S.,Camaschella,C.,Iolascon,A.,andGros,P.(2006)BloodCellsMol.Dis.36,347–354
40.Vidal,S.M.,Pinner,E.,Lepage,P.,Gauthier,S.,andGros,P.(1996)J.Im-
munol.157,3559–3568
41.Ohno,H.,Stewart,J.,Fournier,M.C.,Bosshart,H.,Rhee,I.,Miyatake,S.,Saito,T.,Gallusser,A.,Kirchhausen,T.,andBonifacino,J.S.(1995)Sci-
Nramp1LysosomalTargetingMotif
OCTOBER20,2006•VOLUME281•NUMBER42JOURNALOFBIOLOGICALCHEMISTRY31687
byguestonApril3,2018
http://www.jbc.org/
Downloadedfrom
ence269,1872–1875
42.Boll,W.,Ohno,H.,Songyang,Z.,Rapoport,I.,Cantley,L.C.,Bonifacino,J.S.,andKirchhausen,T.(1996)EMBOJ.15,5789–5795
43.Rohrer,J.,Schweizer,A.,Russell,D.,andKornfeld,S.(1996)J.CellBiol.
132,565–576
44.Guarnieri,F.G.,Arterburn,L.M.,Penno,M.B.,Cha,Y.,andAugust,J.T.(1993)
J.Biol.Chem.268,1941–1946
45.Honing,S.,Griffith,J.,Geuze,H.J.,andHunziker,W.(1996)EMBOJ.15,
5230–5239
46.Ohno,H.,Fournier,M.C.,Poy,G.,andBonifacino,J.S.(1996)J.Biol.
Chem.271,29009–29015
47.Harter,C.,andMellman,I.(1992)J.CellBiol.117,311–325
Nramp1LysosomalTargetingMotif
31688JOURNALOFBIOLOGICALCHEMISTRYVOLUME281•NUMBER42•OCTOBER20,2006
byguestonApril3,2018
http://www.jbc.org/
Downloadedfrom
StevenLam-Yuk-Tseung,VirginiePicardandPhilippeGros
Nramp1(Slc11a1)ThatIsImportantforLysosomalTargeting
IdentificationofaTyrosine-basedMotif(YGSI)intheAminoTerminus
of
doi:10.1074/jbc.M601828200originallypublishedonlineAugust12,2006
2006,281:31677-31688.
J.Biol.Chem.
10.1074/jbc.M601828200
Accessthemostupdatedversionofthisarticleatdoi:
Alerts:
Whenacorrectionforthisarticleisposted
•
Whenthisarticleiscited
•
tochoosefromallofJBC’se-mailalerts
Clickhere
http://www.jbc.org/content/281/42/31677.full.html#ref-list-1Thisarticlecites47references,28ofwhichcanbeaccessedfreeat
byguestonApril3,2018
http://www.jbc.org/
Downloadedfrom